title: Characterization of helical cleavages in type II collagen generated by matrixins.
abstract: Several vertebrate collagenases have been reported to cleave type II collagen, leading to irreversible tissue destruction in osteoarthritis. We have investigated the action of MMP-1 and MMP-13 on type II collagen by use of neoepitope antibodies and N-terminal sequencing. Previous studies have suggested that the initial cleavage of type II collagen by MMP-13 is followed by a second cleavage, three amino acids carboxy-terminal to the primary cleavage site. We show here that this cleavage is also produced by APMA-activated MMP-1 in combination with MMP-3 (i.e. fully activated MMP-1). The use of a selective inhibitor of MMP-3 has shown that it is this enzyme, rather than interstitial collagenase which had been exposed to MMP-3, which makes the second cleavage. In addition we have identified, through N-terminal sequencing, a third cleavage site, three residues carboxy-terminal to the secondary site. Since MMP-2 is thought to be responsible for gelatinolytic action on type II collagen we have investigated the effect of MMP-2 after the initial helical cleavage made by either MMP-1 or MMP-13. A combination of MMPs-1, -2 and -3 results in both the second and third cleavage sites; adding MMP-2 to MMP-13 did not alter the cleavage pattern produced by MMP-13 on its own. We conclude that none of the three cleavage sites will provide information about the specific identity of the collagenolytic enzymes involved in collagen cleavage in situ. Staining of cartilage sections of osteoarthritis patients with the neoepitope antibodies revealed type II collagen degradation starting at or near the articular surface and extending into the mid and deep zones with increasing degeneration of the cartilage.
authors.affiliation: Institute for Bone & Joint Medicine, Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, U.K.
authors.forename: M
authors.initials: M
authors.lastname: Vankemmelbeke
authors.forename: P M
authors.initials: PM
authors.lastname: Dekeyser
authors.forename: A P
authors.initials: AP
authors.lastname: Hollander
authors.forename: D J
authors.initials: DJ
authors.lastname: Buttle
authors.forename: J
authors.initials: J
authors.lastname: Demeester
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002417:Cattle; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D020781:Matrix Metalloproteinase 1; D053509:Matrix Metalloproteinase 13; D010003:Osteoarthritis / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011817:Rabbits
chemical_list: D000906:Antibodies; D007106:Immune Sera; D010446:Peptide Fragments; D003094:Collagen; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D020781:Matrix Metalloproteinase 1